Allergic diseases are still a major health problem due to their high incidence (20 to 30% of the population) and to the lack of curative treatment. Usually the therapy is restricted to the use of antihistamines or to more or less effective immunization procedures. The classical antiallergic drugs have certain disadvantages, especially since they cause various side effects in the treated patient. The immunization procedure is limited to one or two allergens whereas most of the patients are sensitive to a large number of allergens. In addition, hyposensitization treatment is neither curative nor protective.
The vast majority of allergic diseases are mediated by immunoglobulin E (IgE) antibodies directed against a myriad of airborne allergens, e.g. pollens, animal danders, dust mite, food antigens, pharmacological agents, e.g. penicillins, or hymenoptera venom. The mechanisms regulating the production of IgE have been extensively investigated in laboratory animals [K. Ishizaka, Ann. Rev. Immunol. 2, 159 (1984)]. These studies have clearly indicated the existence of non-antigen specific but IgE isotype specific mechanisms controlling the production of IgE in animal models. The effector molecules of these regulatory mechanisms were named IgE-binding factors (IgE-BFs) owing to their affinity for IgE. IgE-BFs may be divided into IgE-suppressive factors (IgE-SFs) and IgE-potentiating factors (IgE-PFs): These molecules differ only by their carbohydrate content. IgE-SFs are not glycosylated or less glycosylated than the corresponding IgE-PFs. The actual production of IgE in animal models is determined by the ratio between these two kinds of IgE-BFs.
The same cells are capable of secreting either IgE-SFs or IgE-PFs depending on the influence of either glycosylation inhibiting or enhancing factors which are secreted by distinct regulatory T lymphocyte subpopulations.
M. Sarfati et al. [Immunology 53, 197, 207, 783 (1984)] have documented the existence of human B cell lines secreting IgE-BFs endowed with similar biological activities as those described in rodents. Other investigators have described the production of IgE-BFs by human T cells [T. F. Huff and K. Ishizaka, Proc. Natl. Acad. Sci. (U.S.A) 81, 1514 (1984)] and by genetic engineering methods [European Patent Application 155 192]. The relationships between IgE-BFs of T cell origin and those of B cell origin were not known hitherto. As will become evident from the present invention the IgE-BF of B-cell origin has less than statistical homology with the IgE-BF of T-cell origin.
Purified IgE-BFs are important for the diagnosis and therapy of allergic diseases and immune regulation diseases connected therewith. In particular IgE-BFs with IgE-suppressive activity might be useful in the treatment of allergic diseases, whereas IgE-BFs with IgE-potentiating activity might increase resistance to infections, for example resistance to parasitic infections.
Assays for the detection of IgE-BFs from B cells are based on a rosette inhibition test wherein RPMI 8866 cells (a lymphoblastoid B cell line) expressing receptors for IgE (Fc.sub..epsilon. R) are rosetted with IgE-coated bovine erythrocytes. If the latter are first preincubated with IgE-BFs, they are no longer able to bind to RPMI 8866 cells and the proportion of cells forming rosettes is reduced accordingly. This assay is not quantitative, it is technically delicate due to the variability in the coupling of IgE to bovine erythrocytes and it is cumbersome, because rosettes must be examined under the microscope, cell lines must be permanently available, IgE-coated erythrocytes must be prepared regularly, etc., so that only a small number of tests (20-40) can be performed reasonably by one person in one day. In a more convenient, quantitative and easy to perform assay monoclonal antibodies to lymphocyte Fc.sub..epsilon. R which are crossreacting with IgE-BFs are utilised. Such monoclonal antibodies have been prepared by G. Delespesse et al., EP 86810244.3, and the hybridoma cell lines producing them are deposited at the Collection Nationale de Cultures de Microorganismes, Institute Pasteur, Paris and are available under accession No. I-425 (clone 208.25 A. 4,3/135), I-420 (clone 208.25 D. 2,1/176), I-451 (clone 207.25 A. 4,4/30), I-452 (clone 207.25 A.4,4/45), and I-486 (clone 208.25 D. 2/94). The corresponding monoclonal antibodies are named Mab-135, Mab-176, Mab-30, Mab-45 and Mab-94, respectively. They allow also an efficient purification of IgE-BFs by affinity chromatography.
Despite the use of above monoclonal antibodies it was hitherto impossible to determine the amino acid sequence of a single IgE-BF isolated from a natural human B-cell line. For therapeutic purposes it would be highly desirable to have a clean, single polypeptide with a defined amino acid sequence having the desired IgE-binding property, and which can be easily prepared in large amounts.
The fast progress in recombinant DNA methods in recent years provides the general methods for achieving this goal. In cases, where the sturcture of the polypeptide is unknown, the sucess of the recombinant DNA technique depends on identification of a mRNA or a DNA coding for the desired polypeptide from a natural source.
After identification of a mRNA by means of a suitable assay, a complementary DNA can be prepared. The cDNA can be incorporated into a suitable vector, whereupon transformation of a suitable host with the obtained hybrid vector, selection and culturing of the transformed hosts allows production and finally isolation of the polypeptide. Isolation of the cDNA coding for the desired polypeptide and sequencing allows determination of the amino acid sequence of the polypeptide. The cDNA or parts thereof can be used to screen the mRNA or the DNA genome of the natural source for further nucleotide sequence coding for the desired polypeptides.
Accordingly, in the present invention, as the structures of the IgE-BFs were unknown, the first objective was to identify a mRNA of human B-cells coding for the desired polypeptide by transforming eggs of Xenopus laevis with fractioned mRNA of said B-cells and determining the clones containing the desired mRNA by an assay utilizing the above monoclonal antibodies. Further objectives were the preparation of cDNAs, and hybrid vectors, the transformation of suitable hosts and finally culturing said hosts and isolating the desired polypeptides. The latter are not necessarily identical with the naturally occurring polypeptides because posttranslational modifications can take place after expression of the polypeptide.